Various binding assays using an immobilized or an immobilizable member of a specific binding pair have been proposed which accomplish the desired separation of a bound and a free form of a labeled reagent. In particular, a number of such binding assays have been described wherein an antibody to an antigen to be detected is bound to an immobilizing material such as the inner wall of a test tube or a plastic bead. For example, U.S. Pat. No. 4,243,749 discloses a competitive binding assay wherein a reaction is carried out in a test tube having a specific antibody to a hapten under determination insolubilized or immobilized on the inner wall of the test tube. The reaction includes a labeled hapten conjugate wherein the quantity of the labeled hapten conjugate, which becomes bound to the test tube wall, is inversely proportional to the amount of the hapten under determination. Similarly, U.S. Pat. No. 4,230,683 discloses a method employing a polystyrene bead having antigen or antibody bound thereto wherein the antigen or antibody is reacted with a hapten-conjugated antibody to the antigen or antibody. The bound hapten-conjugated antibody is further reacted with labeled anti-hapten antibody in order to determine the mount of antigen or antibody in a test sample.
Immobilized catalytically-active-molecules such as enzymes have been used as binding participants to determine the presence or amount of the immobilized enzyme's substrate that may be present in a test sample. Commercial chemistry analyzers use enzymes, which have been bound to porous surfaces, for the conversion of enzyme substrates to optically or electrochemically detectable products. Optical analyzers have been described which have tubular nylon flow cells having enzyme immobilized to the inner walls of the tubing. Optical analyzers which have flow cells containing chromatographic particles (eg, microporous glass) having enzyme immobilized thereon have also been described.
Electrochemical sensors have been described which utilize the ability of an immobilized enzyme to form an electrochemically active molecule as a result of the action of the enzyme on its substrate. Such sensors include certain amperometric electrodes that consist of an enzyme electrode, a reference electrode, a counter electrode and a potentiostat. The enzyme electrode is typically made of platinum and is supplied with an overlaying oxidase enzyme layer. When the electrode is immersed in a sample containing an oxidizable substrate and molecular oxygen, both molecules diffuse into the enzyme layer where the substrate reacts with the enzyme which results in reduction of the enzyme. The reduced enzyme is oxidized by the molecular oxygen which, in turn, is reduced to peroxide. At a sufficiently high electrode potential (measured via the reference electrode), the platinum portion of the enzyme electrode oxidizes the peroxide to regenerate the oxygen and produce two free electrons. The free electrons flow from the enzyme electrode to the counter electrode which completes the circuit consisting of the three electrodes. The potentiostat, which is connected to the circuit, is able to measure the current generated by the electrons and the current measured is related to the amount of oxidizable substrate in the sample. Hence, the presence and/or amount of an oxidizable substrate in the sample can be determined.
Currently available amperometric electrodes which have enzyme electrodes suffer limitations associated with their manufacture. These limitations are generally traceable to the expensive components used to manufacture such electrodes and/or the complex processes used to manufacture them.
For example, U.S. Pat. No. 4,987,075 discloses an enzyme electrode comprising a platinum electrode having a layer of enzymes, which are bound to cellulose with isocyanate monomers and polyurethane polymers, applied thereon. However, the enzyme electrode requires a separate platinum electrode to complete the amperometric electrode.
The immobilization of enzymes directly onto electrode surfaces has also been described. For example, the immobilization of enzymes on platinum surfaces using synthetic polymers has been described (Great Britain Patent Number 2,230,865); sulfonated polyester polymers containing entrapped glucose oxidase has also been used to coat platinum surfaces (Analytic Chimica Acta, 245, 139-143 (1991)); physically entrapped enzyme in gamma irradiated polyvinyl alcohol matrices on platinized graphite electrodes has been described (Biosensors and Bioelectronics, 5, 47-61 (1990)); a glucose biosensor electrode in which enzyme is incorporated in a silicone water based elastomer has been described (U.S. Pat. No. 4,938,860); and a multilayer microfabrication process by which enzyme electrodes can be manufactured on a silicon wafer has been described (U.S. Pat. No. 5,200,051). However, as mentioned above, such electrodes are expensive and unsuited for mass production.
Another method of manufacturing an enzyme electrode incorporates enzymes into a matrix containing both the electrode material and the enzyme (Anal. Chem., 62, 318-320 (1990)). However, the electrode produced in the manner therein described, must be periodically regenerated by polishing the electrode to expose fresh enzyme.
A further method for manufacturing enzyme electrodes has been described in U.S. Pat. No. 5,160,418. The electrode therein described is made with a suspension consisting of an enzyme adsorbed to finely divided platinized carbon which is added to a low temperature binding agent such as gelatin or hydroxyethyl cellulose. The suspension is then deposited on an electrically conductive carbon paper and allowed to dry. However, the binding agents are water soluble and, as a result, the suspension dissolves when it is immersed in an aqueous sample. Hence the electrode is not reusable.